Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell-embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo-reversible hydrogel (TR-HG), based on a modified polysaccharide with a thermo-reversible polyamide [poly(N-isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain-induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR-HG; hMSCs were additionally preconditioned with rhGDF-5 for 7 days. Then, TR-HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR-HG (material control); (D4) PDDM + TR-HG + bNPCs; (D5) PDDM + TR-HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel-filled PDDM and assessment of disc height and volume changes. In gel-injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load-bearing hydrogels that are also cyto-compatible.

A retroperitoneal bronchogenic cyst mimicking a pancreatic or adrenal mass

Retroperitoneal location of bronchogenic cysts is extremely rare. Most commonly they are encountered in the posterior mediastinum. Bronchogenic cysts arise from developmental aberrations of the tracheobronchial tree in the early embryologic period. We report a 42-year-old female patient with a retroperitoneal bronchogenic cyst in the left adrenal region. She was admitted to our hospital with epigastric pain and subsequently underwent CT of the abdomen. The examination revealed a mass related to the left adrenal gland. Endocrine tests for adrenal hypersecretion were negative. Because of the uncertain entity, laparoscopic adrenalectomy was performed. Pathological examination revealed a bronchogenic cyst in proximity to an inconspicuous left adrenal gland. Although very rare, bronchogenic cysts should be considered in the differential diagnosis of retroperitoneal cystic lesions and surgical resection pursued for symptom resolution and to establish a definitive histology.

Eemian and Holocene sea-surface conditions in the southern Black Sea: organic-walled dinoflagellate cyst record from core 22-GC3

In order to compare the sea-surface conditions in the Black Sea during the Holocene and Eemian, sapropelic parts of marine core 22-GC3 (42°13.53′N/36°29.55′E, 838 m water depth) were studied for organic-walled dinoflagellate cyst content. The record shows a change from freshwater/brackish assemblages (Pyxidinopsis psilata, Spiniferites cruciformis, and Caspidinium rugosum) to more marine assemblages (Lingulodinium machaerophorum and Spiniferites ramosus complex) during each interglacial, due to the inflow of saline Mediterranean water. The lacustrine–marine transitions in 22-GC3 occurred at ~ 8.3 cal kyr BP during the early Holocene and ~ 128 kyr BP during the early Eemian, slightly later compared to the onset of interglacial conditions on the adjacent land. Dinoflagellate cyst assemblages reveal higher sea-surface salinity (~ 28–30) (e.g. Spiniferites pachydermus, Bitectatodinium tepikiense, and Spiniferites mirabilis) around ~ 126.5–121 kyr BP in comparison to the Holocene (~ 15–20) as well as relatively high sea-surface temperature (e.g. Tuberculodinium vancampoae, S. pachydermus, and S. mirabilis) especially at ~ 127.6–125.3 kyr BP. Establishment of high sea-surface salinity during the Eemian correlates very well with reconstructed relatively high global sea-level and is explained as a combined effect of increased Mediterranean supply and high temperatures at the beginning of the last interglacial. The observed changes in the dinocyst record highlight the importance of nutrients for the composition of the Eemian and Holocene dinocyst assemblages.

Functional and anatomical outcome of eyes with neovascular age-related macular degeneration treated with intravitreal ranibizumab following an exit strategy regimen.

AIMS To assess the functional and morphological outcome of eyes with neovascular AMD treated with intravitreal ranbizumab following an exit strategy treatment regime. METHODS The Bern treatment regime for neovascular AMD has a fixed injection schedule, even in the non-active stage of the disease. The regimen has been adapted from the PIER study treatment protocol. Eyes with non-active AMD will receive 4 injections in the first year, and 2 injections in the second year of follow-up before treatment stops. Patients that received ranibizumab for treatment and reached the exit criteria were identified, and charts were reviewed to assess functional and morphological outcome. RESULTS Only 2.6% of all patients (15 out of 575 patients) reached the exit criteria. Mean change in best corrected ETDRS visual acuity (VA) was 4.5±16.9 letters when comparing baseline VA to 4 weeks after the last injection (p=0.32). OCT mean foveal thickness was significantly thinner after last treatment (247.9±43.0 µm) compared to baseline (332.5±83.1 µm, p=0.002). The mean total number of ranibizumab injections was 15.6±8.0, and the mean total treatment period was 40.9±18.3 months. Twenty percent of eyes had geographic atrophy present at baseline versus 46.6% at the end of treatment. CONCLUSIONS Even with a fixed treatment regime and a defined treatment exit strategy, only a small percentage of patients reach exit criteria. Retinal thickness has been significantly reduced by repeated intravitreal ranibizumab injections, and geographic atrophy became more frequent.

Methylnitrosourea (MNU)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research. A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes. In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Diagnosis and interdisciplinary treatment of a botryoid odontogenic cyst in the posterior mandible: report of a case.

Botryoid odontogenic cysts (BOC) are considered to be rare polycystic variants of lateral periodontal cysts characterized by a multilocular growth pattern. The most frequent location of BOC is the mandible, predominantly the premolar-canine area, followed by the anterior region of the maxilla. The cyst shows a slight female predilection. This case report of a BOC demonstrates a treatment with initial fenestration and decompression of the cyst in order to prevent damage to adjacent structures such as the inferior alveolar nerve. The present case report emphasizes the importance of accurate clinical, radiographic, and histologic diagnostic procedures of unspecific radiolucent lesions in the jaws to establish a firm diagnosis and avoid inappropriate treatment strategies.

Compressive strength of elderly vertebrae is reduced by disc degeneration and additional flexion

Computer tomography (CT)-based finite element (FE) models assess vertebral strength better than dual energy X-ray absorptiometry. Osteoporotic vertebrae are usually loaded via degenerated intervertebral discs (IVD) and potentially at higher risk under forward bending, but the influences of the IVD and loading conditions are generally overlooked. Accordingly, magnetic resonance imaging was performed on 14 lumbar discs to generate FE models for the healthiest and most degenerated specimens. Compression, torsion, bending, flexion and extension conducted experimentally were used to calibrate both models. They were combined with CT-based FE models of 12 lumbar vertebral bodies to evaluate the effect of disc degeneration compared to a loading via endplates embedded in a stiff resin, the usual experimental paradigm. Compression and lifting were simulated, load and damage pattern were evaluated at failure. Adding flexion to the compression (lifting) and higher disc degeneration reduces the failure load (8–14%, 5–7%) and increases damage in the vertebrae. Under both loading scenarios, decreasing the disc height slightly increases the failure load; embedding and degenerated IVD provides respectively the highest and lowest failure load. Embedded vertebrae are more brittle, but failure loads induced via IVDs correlate highly with vertebral strength. In conclusion, osteoporotic vertebrae with degenerated IVDs are consistently weaker—especially under lifting, but clinical assessment of their strength is possible via FE analysis without extensive disc modelling, by extrapolating measures from the embedded situation.

Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study

PURPOSE To evaluate the safety, tolerability and bioactivity of ascending doses of MP0112, a designed ankyrin repeat protein (DARPin) that binds with high affinity to vascular endothelial growth factor-A (VEGF-A), in treatment-naive patients with exudative age-related macular degeneration (AMD). DESIGN Phase I/II, open-label, multicenter, dose-escalation study. METHODS Patients were to receive a single intravitreal injection of MP0112 at doses ranging from 0.04 to 3.6 mg and be monitored for 16 weeks for safety, efficacy, pharmacokinetics, and dose response. RESULTS Altogether, 32 patients received a single injection of MP0112. The maximum tolerated dose was 1.0 mg because of a case of endophthalmitis in the 2.0 mg cohort. Drug-related adverse events were reported by 13 (41%) of 32 patients; they included ocular inflammation in 11 patients (7 mild, 4 moderate in severity). Visual acuity scores were stable or improved compared with baseline for ≥4 weeks following injection; both retinal thickness and fluorescein angiography leakage decreased in a dose-dependent manner. Rescue therapy was administered to 20 (91%) of 22 patients who received 0.04-0.4 mg MP0112 compared with 4 of 10 (40%) patients who received 1.0 or 2.0 mg. Of patients in the higher-dose cohorts who did not require rescue treatment, 83% (5/6) maintained reductions in central retinal thickness through week 16. CONCLUSIONS A single injection of 1.0 or 2.0 mg MP0112 resulted in mean decreases in retinal thickness and leakage area despite ocular inflammation. Larger-scale studies are warranted to confirm these observations.

Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy.

In recent decades the application of bioreactors has revolutionized the concept of culturing tissues and organs that require mechanical loading. In intervertebral disc (IVD) research, collaborative efforts of biomedical engineering, biology and mechatronics have led to the innovation of new loading devices that can maintain viable IVD organ explants from large animals and human cadavers in precisely defined nutritional and mechanical environments over extended culture periods. Particularly in spine and IVD research, these organ culture models offer appealing alternatives, as large bipedal animal models with naturally occurring IVD degeneration and a genetic background similar to the human condition do not exist. Latest research has demonstrated important concepts including the potential of homing of mesenchymal stem cells to nutritionally or mechanically stressed IVDs, and the regenerative potential of "smart" biomaterials for nucleus pulposus or annulus fibrosus repair. In this review, we summarize the current knowledge about cell therapy, injection of cytokines and short peptides to rescue the degenerating IVD. We further stress that most bioreactor systems simplify the real in vivo conditions providing a useful proof of concept. Limitations are that certain aspects of the immune host response and pain assessments cannot be addressed with ex vivo systems. Coccygeal animal disc models are commonly used because of their availability and similarity to human IVDs. Although in vitro loading environments are not identical to the human in vivo situation, 3D ex vivo organ culture models of large animal coccygeal and human lumbar IVDs should be seen as valid alternatives for screening and feasibility testing to augment existing small animal, large animal, and human clinical trial experiments.